Detection reagent for detecting AD target Micro RNA, application of detection reagent and detection kit

Abstract

The invention belongs to the technical field of in-vitro detection, and particularly relates to a detection reagent for detecting AD target Micro RNA, application of the detection reagent and a detection kit. According to the present invention, the AD disease detection target is screened, the concentration, the reaction temperature and the reaction time of each component in the detection system are optimized, and the CHA-CRISPR detection reagent is delivered by using the liposome to prepare the detection kit, such that the high-sensitivity, high-specificity and high-accuracy detection of the MicroRNA in the AD patient exosome is achieved, and the advantages of simpleness, convenience and rapidness are provided. The invention provides a rapid and efficient detection method for Alzheimer's disease detection, and the application prospect is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro detection, and specifically relates to a detection reagent for detecting AD target Micro RNA, and a use thereof and a detection kit. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative dementia characterized by progressive cognitive decline in memory, orientation, language, behavior, etc. Early diagnosis and intervention are considered to be important means to slow the progression of the disease. Common detection methods for AD include neuroimaging and cerebrospinal fluid (CSF) biomarker detection. The former uses "carbon 11" markers to detect brain β-amyloid protein (Aβ) deposits, but it is expensive and requires professional instruments and personnel, and is not suitable for screening. In the latter, β-amyloid protein and tau protein in cerebrospinal fluid are recognized AD biomarkers with high diagnostic accuracy. However, cerebrospinal fluid collection is an invasive operation, which requires high operator requirements and is not suitable for screening and routine testing.

[0003] Liquid biopsy is a non-invasive diagnostic and predictive tool that collects and analyzes body fluids (such as blood, urine, and saliva) to obtain disease-related information. Exosomes (EVs) are extracellular vesicles secreted by cells with a diameter of <200 nm. The cells contain nucleic acids, lipids, proteins and other substances from the source cells. Studies have shown that the abnormal expression of MicroRNA is closely related to the occurrence and development of diseases. Unlike the easily degradable MicroRNA exposed in body fluids, the MicroRNA carried in exosomes is stable.

[0004] At present, the traditional detection of biological molecules contained in EVs requires the steps of lysing the enriched EVs, extracting the RNA and proteins therein, and then conducting subsequent detection and analysis processes. This will interfere with the identification of biological molecular signals of EVs derived from specific tissue cells, and there is also the risk of biological molecule loss.

[0005] Liposomal vesicles are artificial microbubble structures that simulate carrying functional substances in a limited space. They can fuse with EVs of the same or similar biochemical components to ensure the integrity of the membrane. However, the concentration of MicroRNA in exosomes is extremely low, so nucleic acid amplification technology is also needed to achieve signal amplification.

[0006] PCR is the most common nucleic acid amplification technology, but this technology requires multiple raw materials such as primers, Taq polymerase, dNTPs, and professional instruments to achieve repeated temperature changes for amplification. Not only is the experimental process unpredictable, it will also damage EVs and put RNA at risk of contamination or degradation. Constant temperature nucleic acid amplification technology can ensure the integrity of EVs, but non-specific amplification often leads to false positive results. CRISPR technology combined with constant temperature nucleic acid amplification technology can achieve a highly sensitive and specific nucleic acid detection method, but the reaction process needs to be carried out step by step. Because amplification and shearing are intertwined, high requirements are placed on reaction reagents, and the experimental results are also affected.

[0007] Based on the above problems, there is an urgent need to develop a MicroRNA molecular detection technology that does not require enzymes, is simple to operate, and has high sensitivity and specificity to provide support for the early diagnosis of diseases such as AD. Summary of the invention

[0008] In view of the above problems, the present invention provides a detection reagent for detecting AD target Micro RNA, its use and a detection kit.

[0009] The present invention provides a detection reagent for detecting AD target Micro RNA, characterized in that it comprises the following components in molar parts: H1 25-100 copies, H2 25-100 copies, 10-20 copies of activator, CRISPR / Cas endonuclease 50-125 copies, gRNA 50-100 copies, Report molecules 200-1000 copies; The H1 and H2 are hairpin probe 1 and hairpin probe 2 in the catalytic hairpin self-assembly combined with CRISPR detection method.

[0010] Preferably, the detection reagent for detecting AD target Micro RNA comprises the following components in molar parts: H1 50-75 copies, H2 50 parts, Activator 20 copies, CRISPR / Cas endonucleases 75 copies, gRNA 75 copies, 800 copies of the reporter.

[0011] Preferably, the CRISPR / Cas endonuclease is selected from Cas12a, Cas13a, and Cas9.

[0012] Preferably, the combination of H1 and H2 includes at least one of the following combinations: The nucleotide sequence of H1 is SEQ ID NO. 2, and the nucleotide sequence of H2 is SEQ ID NO. 3; Or, the nucleotide sequence of H1 is SEQ ID NO. 8, and the nucleotide sequence of H2 is SEQ ID NO. 9; Or, the nucleotide sequence of H1 is SEQ ID NO. 11, and the nucleotide sequence of H2 is SEQ ID NO.12.

[0013] Preferably, the nucleotide sequence of gRNA is SEQ ID NO. 4; And / or, the activator is composed of nucleic acids having a nucleotide sequence of SEQ ID NO. 5 and a nucleotide sequence of SEQ ID NO. 6; And / or, the nucleotide sequence of the reporter molecule is TTATT, and both ends of the reporter molecule are modified with a fluorescent group and a fluorescent quenching group, respectively; And / or, H1, H2, and the activator are prepared by dissolving nucleic acid in a buffer solution and subjecting the nucleic acid to thermal annealing treatment; And / or, the detection reagent further comprises a 36-50% buffer, the components of the buffer comprising 36.65-50 mM Tris-HCl, 73.3-100 mM NaCl, 7.33-10 mM MgCl2, 73.3-100 μg / mL BSA, 7.33-10 μg / mL heparin sodium, pH 7.5-8.0.

[0014] The present invention provides the use of any of the above-mentioned detection reagents for detecting AD target Micro RNA, and the use of the detection reagents in preparing a detection kit or a microfluidic chip for MicroRNA in the serum of patients with Alzheimer's disease or cancer.

[0015] Preferably, the method for using the detection kit for MicroRNA in the serum of patients with Alzheimer's disease or cancer comprises the following steps: The test solution is mixed with the detection reagent in the detection kit for detecting MicroRNA in the serum of patients with Alzheimer's disease or cancer, incubated at 37° C. for 30-180 minutes, and the fluorescence signal is detected.

[0016] The invention provides a detection kit, comprising any of the above detection reagents for detecting AD target Micro RNA, or lipid spheres containing any of the above detection reagents for detecting AD target Micro RNA; In the lipid sphere containing any of the above detection reagents for detecting AD target Micro RNA, the dosage ratio of the detection reagent for detecting AD target Micro RNA to the lipid sphere is 1-2 mL: 23-46 mg.

[0017] Preferably, the lipid sphere containing any of the above detection reagents for detecting AD target Micro RNA is prepared according to the following steps: the detection reagent for detecting AD target Micro RNA is mixed with the lipid sphere.

[0018] Preferably, the lipid sphere is prepared according to the following steps: mixing cationic lipid molecules, amphiphilic phospholipid molecules and cholesterol, rotary evaporating to obtain a lipid film, and then incubating and extruding in an aqueous solution to obtain a lipid film; And / or, the mixing is incubated at 37° C. for 6-12 h.

[0019] Preferably, the cationic lipid molecule is (2,3-dioleoyl-propyl)-trimethylammonium-chloride), the amphiphilic phospholipid molecule is (2-oleoyl-1-palmitin glycerol-3-phosphocholine); the mass ratio of the cationic lipid molecule to the amphiphilic phospholipid molecule and cholesterol is 10:10:3.

[0020] The present invention provides a detection reagent for detecting AD target Micro RNA, its use and detection kit. The present invention screens the detection target of AD disease, optimizes the concentration, reaction temperature and reaction time of each component in the detection system, and uses liposomes to deliver the CHA-CRISPR detection system, thereby realizing high sensitivity, high specificity and high accuracy detection of MicroRNA in exosomes of AD patients, which has the advantages of simplicity and rapidity. The present invention provides a fast and efficient detection method for the detection of Alzheimer's disease, and has good application prospects.

[0021] The beneficial effects of the present invention include: (1) The detection system is delivered to the serum to be tested using liposomes, which avoids possible RNA degradation or cross-contamination in conventional detection methods, improves the sensitivity of MicroRNA detection, and provides a more stable and efficient method for identifying exosomal MicroRNA, providing a rapid and efficient method for diagnosing Alzheimer's disease.

[0022] (2) The combination of catalytic hairpin self-assembly (CHA) and CRISPR / Cas technology (CHA-CRISPR) has significant complementarity and innovation, bringing many beneficial effects, especially in the fields of molecular detection, gene editing and diagnosis. First, CHA is a nucleic acid amplification technology, and the trans-cleavage of CRISPR / Cas is a signal amplification technology. The combination of CHA and CRISPR technology realizes the integration of signal amplification capabilities. Second, it improves the sensitivity of detection. CHA can effectively amplify a small number of targets through autocatalytic cycles. After accurately identifying the target DNA or RNA, CRISPR can activate the nuclease activity of Cas proteins (such as Cas12a or Cas13) to further amplify the signal. Third, it makes the detection system have high specificity and low background noise. CRISPR / Cas technology itself has extremely high targeting specificity, and the hairpin structure of CHA is stable, which avoids a lot of background noise. Fourth, it achieves lower costs and simplified operations. CHA does not require expensive temperature-changing equipment or enzymes and other reagents, making the entire detection process cheaper and more convenient, suitable for resource-limited areas, and meeting the needs of low-cost diagnosis. Fifth, CHA combined with CRISPR / Cas technology enhances resistance to background interference. Both CHA and CRISPR / Cas require specific target recognition and are highly stable, and can stably and accurately detect target nucleic acids even in complex samples (such as blood, urine, environmental samples, etc.). Sixth, this method does not require expensive equipment and time-consuming steps and can be used for convenient on-site diagnosis. Seventh, this method has a wide range of applications and can innovate across fields, such as medical diagnosis, gene editing verification, and environmental / food safety monitoring.

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the verification result diagram of the CHA-CRISPR detection system.

[0026] Figure 2 These are the results of the CHA-CRISPR detection system detecting MicroRNA-135a, MicroRNA-193b, and MicroRNA-384 respectively.

[0027] Figure 3It is a screening result diagram of the component ratio in the CHA-CRISPR detection system; wherein A is a screening result diagram of the ratio of H1 to H2, wherein B is a screening result diagram of the ratio of Cas12a to H2, wherein S / N is the sample fluorescence signal / negative control fluorescence signal.

[0028] Figure 4 It is a screening result diagram of the component ratio in the CHA-CRISPR detection system; A is the screening result diagram of the ratio of Cas12a to gRNA, and B is the screening result diagram of the Reporter concentration.

[0029] Figure 5 It is a screening result diagram of reaction temperature and reaction time of CHA-CRISPR detection system; wherein A is the screening result diagram of reaction temperature, and wherein B is the screening result diagram of reaction time.

[0030] Figure 6 The results of Western blot analysis were used to determine the protein contents of CD9, CD63, and CD81 in cells and exosomes.

[0031] Figure 7 are biological transmission electron microscopy images; A is a biological transmission electron microscopy image of exosomes; B is a biological transmission electron microscopy image of the fusion of exosomes and lipid globules.

[0032] Figure 8 These are the results of lipid spheres, exosomes and lipid sphere fusion measured by photodynamic scattering instrument; A is the hydrodynamic diameter result diagram; and B is the zeta potential result diagram.

[0033] Fig. 9 This is a graph showing the results of the detection kit in Example 1 detecting different concentrations of extracellular vesicles in the serum of Alzheimer's patients.

[0034] Fig.10 This is a graph showing the results of the detection kit in Example 1 detecting MicroRNA exosomes from different cells. DETAILED DESCRIPTION

[0035] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0036] Example 1 A detection kit and detection method for detecting AD target MicroRNA-384 1. Composition and preparation method of the kit The kit of this example is prepared by encapsulating the CHA-CRISPR detection system in Table 1 in liposome microspheres.

[0037] (1) The CHA-CRISPR detection system in Table 1.

[0038] Table 1 CHA-CRISPR detection system Note: The components of 1×Buffer are: 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, 100 μg / mL BSA, pH 7.9, 10 μg / mL heparin sodium.

[0039] In other embodiments, in the above CHA-CRISPR detection system, the concentration of H1 can be adjusted in the range of 25-100 nM; the concentration of Cas12a can be adjusted in the range of 50-125 nM; the concentration of gRNA can be adjusted in the range of 25-100 nM; the concentration of Reporter can be adjusted in the range of 200-1000 nM.

[0040] (2) Nucleic acid sequence The nucleic acid sequences involved in this example are shown in Table 2.

[0041] Table 2 Nucleotide sequences Note: The nucleotide sequence of Reporter is TTATT. The 5' end of the Reporter sequence is modified with FAM fluorescent group, and the 3' end is modified with BHQ1 fluorescent quenching group.

[0042] (3) Preparation of nucleic acids H1, H2 and Activator The H1 and H2 chains were incubated in DEPC buffer (pH=7.4) at 95°C for 4 min, and then cooled to 10°C at a rate of 1.5°C / min to form a hairpin structure. Activator was obtained by hybridizing Activator-TS and Activator-NTS using the same method and annealing procedure.

[0043] (4) Preparation of lipid spheres loaded with detection system 10 mg (2,3-dioleoyl-propyl)-trimethylammonium-chloride) DOTAP, 10 mg (2-oleoyl-1-palmitin glycerol-3-phosphocholine) POPC and 3 mg cholesterol were dissolved in 1 mL chloroform, and the chloroform was removed by rotary evaporation to form a lipid film at the bottom of the round-bottom flask. The film was hydrated in 10 mL DPBS (37 ° C, 1 hour), oscillated on a vortex for 1 minute, and then formed lipid microspheres by extruding 21 times with a 0.1 μm polycarbonate membrane. 1 mL CHA-CRISPR detection system was added, mixed thoroughly, and placed in a water bath for incubation at 37 ° C for 12 hours. Finally, centrifuged (6000 rpm, 5 minutes), washed 10 times with DPBS, resuspended in 1 mL DPBS, and stored at 4 ° C.

[0044] 2. Detection Method (1) Detection of MicroRNA-384 using the CHA-CRISPR detection system Take 1 μL of the test solution, mix it with the detection system in Table 1, incubate it at 37°C for 120 min, and detect the fluorescence signal.

[0045] (2) Detection of MicroRNA-384 in peripheral blood EVs using a kit Take the blood to be tested, obtain the EVs solution to be tested by differential centrifugation, take 1 μL of the EVs solution to be tested, mix it with 20 μL of lipid spheres loaded with the detection system prepared in this example, incubate at 37° C. for 135 min, and detect the fluorescence signal.

[0046] The specific steps of the above differential centrifugation are: centrifugation at 3000g for 20 minutes, taking the supernatant, centrifuging the supernatant at 11000g for 30 minutes, and then collecting the supernatant, which is the EVs solution to be tested.

[0047] Example 2 A detection kit and detection method for detecting AD target MicroRNA-135a This embodiment provides a detection kit and a detection method for detecting AD target MicroRNA-135a, which are the same as those in Example 1, except that the primer sequences are different and the detected MicroRNA is different.

[0048] The nucleic acid sequences of H1, H2 and the detected MicroRNA-135a in the CHA-CRISPR detection system of this embodiment are shown in Table 3.

[0049] Table 3 Nucleic acid sequences of MicroRNA-135a and its H1 and H2 Example 3 A detection reagent and detection method for detecting AD target MicroRNA-193b This embodiment provides a detection kit and a detection method for detecting AD target MicroRNA-193b, which are the same as those in Example 1, except that the primer sequences are different and the detected MicroRNA is different.

[0050] The nucleic acid sequences of H1, H2 and the detected MicroRNA-193b in the detection system of this embodiment are shown in Table 4.

[0051] Table 4 Nucleic acid sequences of MicroRNA-193b and its H1 and H2 The technical solution of the present invention is further illustrated by experiments below.

[0052] Experimental Example 1 Verification of CHA-CRISPR Detection System and Optimization of Conditions 1. Experimental Methods (1) Testing system The detection system of this experimental example is shown in Table 5: Table 5 CHA-CRISPR detection system Note: The components of 1×Buffer are: 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, 100 μg / mL BSA, pH 7.9, 10 μg / mL heparin sodium.

[0053] (2) Verification of the testing system The verification experiment was divided into 7 groups. After adding each component, the mixture was incubated at 37°C for 90 min to detect the fluorescence signal. Among them, the nucleic acid sequence of H1 was SEQ ID NO. 2, the nucleic acid sequence of H2 was SEQ ID NO. 3, the Activator was prepared according to the preparation method of the Activator described in Example 1, the nucleic acid sequence of gRNA was SEQ ID NO. 4, and the nucleic acid sequence of Reporter was TTATT. The nucleotide sequence of microRNA was SEQ ID NO. 1, the added volume was 1 μL, and the reaction concentration was 10 4 fM. Components added to each group: ① According to the detection system, add Activator, Cas12a, gRNA, Buffer, Reporter; ② According to the detection system, add H1, Activator, Cas12a, gRNA, Buffer, Reporter; ③ According to the detection system, add H2, Activator, Cas12a, gRNA, Buffer, Reporter; ④ According to the detection system, add H1, H2, Activator, Cas12a, gRNA, Buffer, Reporter; ⑤ According to the detection system, add H1, MicroRNA, Activator, Cas12a, gRNA, Buffer, and Reporter; ⑥ According to the detection system, add H2, MicroRNA, Activator, Cas12a, gRNA, Buffer, and Reporter; ⑦ According to the detection system, add H1, H2, MicroRNA, Activator, Cas12a, gRNA, Buffer, and Reporter; (3) Validation of MicroRNA Detection Four detection systems were constructed, namely MicroRNA-384 group, MicroRNA-193b group, MicroRNA-135a group and negative control group. The nucleic acid sequence of H1 in MicroRNA-384 group is SEQ ID NO. 2, and the nucleic acid sequence of H2 is SEQ ID NO. 3; the nucleic acid sequence of H1 in MicroRNA-193b group is SEQ ID NO. 11, and the nucleic acid sequence of H2 is SEQ ID NO. 12; the nucleic acid sequence of H1 in MicroRNA-135a group is SEQ ID NO. 8, and the nucleic acid sequence of H2 is SEQ ID NO. 9; the nucleic acid sequence of H1 in the negative control group is SEQ ID NO. 2, and the nucleic acid sequence of H2 is SEQ ID NO. 3.

[0054] According to the detection system in Table 5, 1 μL of MicroRNA-135a, MicroRNA-193b, MicroRNA-384 and distilled water were added to the four detection systems respectively, incubated at 37°C for 90 min, and the fluorescence signal was detected.

[0055] (4) Optimization of components in the detection system The condition optimization experiment was divided into 6 groups. According to the detection system in Table 5, the concentration of H1, the concentration of Cas12a, the concentration ratio of cas12a and gRNA, the concentration of Reporter, the reaction temperature and the reaction time were optimized respectively. The nucleic acid sequence of H1 in the detection system is SEQ ID NO. 2, and the nucleic acid sequence of H2 is SEQ ID NO. 3. The target gene detected is 10 4 fM of MicroRNA-384, and 1 μL PBS was added to the detection system for the negative control group.

[0056] The following are the experimental methods for each group: ① When the concentrations of H1 were tested were 25nM (H1:H2=0.5:1), 50nM (H1:H2=1:1), 75nM (H1:H2=1.5:1), and 100nM (H1:H2=2:1), the fluorescence intensity of the target gene at the same concentration was detected under incubation conditions at 37°C for 90 min.

[0057] ② When the concentrations of Cas12a were tested were 25nM (Cas12a:H2=0.5:1), 50nM (Cas12a:H2=1:1), 75nM (Cas12a:H2=1.5:1), 100nM (Cas12a:H2=2:1), and 125nM (Cas12a:H2=2.5:1), the fluorescence intensity of the target gene at the same concentration was detected under incubation conditions of 37°C for 90 min.

[0058] ③ The concentration ratios of cas12a and gRNA tested were 25nM:25nM (Cas12a: gRNA=0.5:0.5), 25nM:50nM (Cas12a: gRNA=0.5:1), 25nM:75nM (Cas12a: gRNA=0.5:1.5), 25 nM:100nM (Cas12a:gRNA=0.5:2), 50 nM:25 nM (Cas12a: gRNA=1:0.5), 50 nM:50 nM (Cas12a:gRNA=1:1), 50 nM:75 nM (Cas12a: gRNA=1:1.5), 50 nM:100 nM (Cas12a: gRNA=1:2), 75 nM:25nM (Cas12a:gRNA=1:0.5), 75 nM:50 Figure 2. Fluorescence intensity of target genes at the same concentration at 37°C for 90 min incubation at 75 nM:75 nM (Cas12a:gRNA=1.5:1), 75 nM:75 nM (Cas12a:gRNA=1.5:1.5), 75 nM:100 nM (Cas12a:gRNA=1.5:2), 100 nM:25 nM (Cas12a: gRNA=2:0.5), 100 nM:50 nM (Cas12a:gRNA=2:1), 100 nM:75 nM (Cas12a: gRNA=2:1.5), 100 nM:100 nM (Cas12a:gRNA=2:2).

[0059] ④ When the concentrations of the test reporter were 200 nM, 400 nM, 600 nM, 800 nM, and 1000 nM, respectively, the fluorescence intensity of the target gene at the same concentration was detected under the condition of incubation at 37°C for 90 min.

[0060] ⑤ When the reaction temperatures were 35°C, 37°C, 39°C, and 42°C, and the incubation time was 90 min, the fluorescence intensity of the target gene at the same concentration was detected.

[0061] ⑥ When the test reaction time was 30 min, 60 min, 90 min, 120 min, 150 min and 180 min respectively, the fluorescence intensity of the target gene with the same concentration was detected under the incubation condition of 37°C.

[0062] 2. Experimental Results (1) Verification of the testing system The verification results of the test system are as follows: Figure 1 As shown: When there is no H1, H2 or target Micro RNA in the detection system, the fluorescence signal is very weak, indicating that the detection system has a low background signal. Only when H1, H2 and target Micro RNA are present at the same time, can a clear fluorescence signal be detected, indicating that H1, H2 interact with the target Micro RNA, resulting in the amplification of the fluorescence signal, proving that the detection system of this experimental example has been verified to be used for the detection of Micro RNA-384.

[0063] (2) Validation of MicroRNA Detection Verification results are as follows Figure 2 As shown: compared with the negative control group, the detection system of this experimental example successfully amplified and detected MicroRNA-384, MicroRNA-193b, and MicroRNA-135a, respectively, and the CHA-CRISPR method of the present invention was successfully established.

[0064] (3) Optimization of components in the detection system Filter results such as Figure 3-5 As shown: Under each screening experimental condition, the enhancement of fluorescence signal was detected. The results of comprehensive detection of signal intensity and signal-to-noise ratio, in particular, when the ratio of H1:H2 was 1:1, the ratio of Cas12a:H2 was 1.5-2.5:1, the concentration ratio of Cas12a:gRNA was 1-1.5:1-1.5, the concentration of Reporter was 800nM, the reaction temperature was 37℃, or the reaction time was 120min, the fluorescence signal enhancement effect was the most obvious, and the signal-to-noise ratio was high.

[0065] The above experiments show that the detection system of the present invention has a low background signal; when H1 and H2 are changed, MicroRNA-135a, MicroRNA-193b, and MicroRNA-384 can be detected accordingly. The preferred conditions of the detection system are: the optimal ratio of H1:H2:Cas12a:gRNA is 1:1:1.5:1:1.5, the optimal concentration of Reporter is 800 nM, the optimal reaction temperature is 37°C, and the optimal reaction time is 120 min.

[0066] Experimental Example 2 Characterization of exosome-lipid sphere fusion 1. Experimental Methods (1) Cell culture Human neuroblastoma (SH-SY5Y cells) and human neural stem cells (hNSCs) were cultured in DMEM containing 10% FBS (without extracellular vesicles), 100 units / mL penicillin, and 100 units / mL streptomycin at 37°C and 5% CO2. To prepare FBS without extracellular vesicles, FBS was ultracentrifuged at 110,000g for 10 h and then filtered with a 0.22 μm filter membrane (Thermo Fisher Scientific, Shanghai) and stored at -20°C.

[0067] (2) Extraction of exosomes ① Collect the cell culture medium and centrifuge at 3000g for 20 minutes to remove cells and large cell debris.

[0068] ② Centrifuge the cell culture medium obtained in the above step at 11000g for 30 minutes to remove small particle cell debris and collect the supernatant.

[0069] ③ Ultracentrifuge the supernatant at 110,000 g for 140 minutes to obtain exosomes (also known as extracellular vesicles).

[0070] ④ The obtained cell exosomes were dissolved in Dulbecco's phosphate buffered saline (DPBS) (purchased from Shanghai Thermo Fisher Scientific Co., Ltd.) and washed by ultracentrifugation at 110,000 g for 140 minutes. Finally, the cell exosomes were resuspended in DPBS and stored at -80°C for further use.

[0071] (3) Fusion of exosomes and lipid globules 10 mg (2,3-dioleoyl-propyl)-trimethylammonium-chloride) DOTAP, 10 mg (2-oleoyl-1-palmitin glycerol-3-phosphocholine) POPC and 3 mg cholesterol were dissolved in 1 mL of chloroform, and the chloroform was removed by rotary evaporation to form a lipid film at the bottom of a round-bottom flask. The film was hydrated in DPBS (37°C, 1 hour), shaken on a vortex for 1 minute, and placed in a water bath for incubation at 37°C for 12 hours after being fully mixed. Then, lipid spheres were prepared by extruding 21 times with a 0.1 μm polycarbonate membrane.

[0072] The lipid spheres and exosomes were incubated at 37°C for 15 min to characterize the fusion of exosomes and lipid spheres.

[0073] (3) Western blot experiment of exosomes ② Western blot analysis of exosome markers (CD9, CD63, CD81): SH-SY5Y cells and exosome sediments were mixed with 200 μL RIPA lysis buffer (Solebo Biotechnology Co., Ltd., Beijing), and the CD9, CD63, and CD81 protein concentrations were determined using the BCA protein detection kit (Solebo Biotechnology Co., Ltd., Beijing), and then denatured at 95°C for 5 min. The loading amount of cells was obtained by the internal reference protein (GAPDH), and the samples were loaded with the specified protein amount to determine whether CD9, CD63, and CD81 were enriched in exosomes compared with cells.

[0074] (4) Transmission electron microscopy characterization of cell exosomes and exosome-lipid sphere fusion The morphology of exosomes and exosome-lipid sphere fusion was characterized by biological transmission electron microscopy (TEM, JEM 1400 Plus, JEOL). The samples were first fixed with 2.5% glutaraldehyde and washed with PBS buffer, then secondarily fixed with 1% osmium acid, washed with PBS buffer, and then dehydrated with ethanol solution. Subsequently, the samples were infiltrated with acetone and epoxy resin overnight, embedded with epoxy resin, and sliced ​​with an ultramicrotome (Leica EM UC7). The slices were placed on copper grids, stained with uranyl acetate, and imaged under a transmission electron microscope at 100 kV.

[0075] (5) Dynamic light scattering characterization of exosomes and fusion of exosomes and lipid globules Dynamic light scattering (DLS) was used to monitor the changes in hydrodynamic diameter during the fusion process of lipid microspheres and exosomes, and the zeta potential of lipid microspheres, exosomes and fused vesicles was measured.

[0076] 2. Experimental Results (1) Western blot characterization results of cell exosomes Western blot results Figure 6 As shown: Compared with cells, CD9, CD63, and CD81 proteins are enriched in exosomes, indicating that exosomes were effectively extracted by the method of this experimental example.

[0077] (2) Transmission electron microscopy results Transmission electron microscopy results of exosomes Figure 7 As shown in A, exosomes are nanometer-sized spheres. The transmission electron microscopy results of exosome fusion with lipid spheres are shown in Figure 7 As shown in B, it can be seen from the figure that the two microspheres are in contact and partially fused, indicating that exosomes and lipid spheres can fuse.

[0078] (3) DLS characterization results DLS results are as follows Figure 8 As shown: The particle size of lipid spheres and exosomes is about 200nm. When the two are fused, the particle size increases significantly, and as time goes by, the size of the fused microspheres gradually increases, indicating that the lipid spheres and exosomes have fused, and as the fusion time increases, the degree of fusion increases. Zeta potential further proves the fusion of lipid spheres and exosomes.

[0079] The above results show that exosomes were extracted from cells in this experimental example, and it was proved that exosomes can fuse with lipid globules.

[0080] Experimental Example 3 Performance Verification of Membrane Fusion / CHA-CRISPR Detection Method The membrane fusion / CHA-CRISPR detection kit and its detection method used in this experimental example are as described in Example 1.

[0081] 1. Sensitivity analysis The concentration of the assay was 10 using the Membrane Fusion / CHA-CRISPR Detection Kit and its assay method. -1 —— 10 5 fM of serum exosomes from patients with Alzheimer's disease, and a negative control was set up, which was distilled water.

[0082] Among them, the concentration of exosomes is measured by the concentration of Micro 384 in the exosomes. The preparation method of exosomes from the serum of Alzheimer's patients with different concentrations is: after collecting the exosomes in the serum of Alzheimer's patients, the concentration of the contained MicroRNA is detected by the PCR method, and after dilution, the exosome solution with the target MicroRNA concentration is obtained.

[0083] Test results such as Fig. 9 As shown in Figure 2, as the concentration of exosomes increases, the fluorescence signal increases. The sensitivity is 10 1 fM, the detection method of Example 1 of the present invention has high sensitivity.

[0084] 2. Specificity Analysis The membrane fusion / CHA-CRISPR detection kit and the detection method thereof were used to detect different types of exosomes, and the different types of exosomes were respectively exosomes isolated from SH-SY5Y cells and hNSCs cells.

[0085] Test results such as Fig.10 As shown, when the exosomes isolated from SH-SY5Y cells were injected, a significantly enhanced fluorescence signal was detected, indicating that the detection method of Example 1 of the present invention has high specificity.

[0086] 3. Clinical Sample Testing Serum from 10 AD patients and 10 healthy subjects was collected and tested using the membrane fusion / CHA-CRISPR detection kit and detection method of Examples 1-3, respectively.

[0087] The test results are shown in Table 6. When MicroRNA-384 is used as the detection target in Example 1, the false negative result is 0 and the false positive result is 20%; when MicroRNA-135a is used as the detection target in Example 2, the false negative result is 20% and the false positive result is 10%; when MicroRNA-193b is used as the detection target in Example 3, the false negative result is 40% and the false positive result is 40%. Therefore, the present invention preferably uses MicroRNA-384 as the detection target.

[0088] Table 6 Clinical sample test results The above results show that the membrane fusion / CHA-CRISPR detection kit and detection method of this embodiment have high sensitivity, specificity and accuracy, can meet the requirements of actual detection, and the preferred detection target is MicroRNA-384.

[0089] Through the above embodiments and experimental examples, it can be seen that the present invention provides a detection reagent, a detection kit and a detection method for detecting AD target Micro RNA. The present invention screens the detection target of AD disease, optimizes the concentration, reaction temperature and reaction time of each component in the detection system, and uses liposomes to deliver the CHA-CRISPR detection system to prepare a detection kit, which achieves high sensitivity, high specificity and high accuracy detection of MicroRNA in exosomes of AD patients, and has the advantages of simplicity and rapidity. The present invention provides a fast and efficient detection method for diagnosing Alzheimer's disease, and has good application prospects.

Claims

1. A detection reagent for detecting AD target Micro RNA, characterized in that: It includes the following components in molar parts: H1 25-100 copies, H2 25-100 copies, 10-20 copies of activator, CRISPR / Cas endonuclease 50-125 copies, gRNA 50-100 copies, Report molecules 200-1000 copies; Said H1 and H2 are hairpin probe 1 and hairpin probe 2 in the catalytic hairpin self-assembly combined with CRISPR detection method; The combination of H1 and H2 includes at least one of the following combinations: The nucleotide sequence of H1 is SEQ ID NO. 2, and the nucleotide sequence of H2 is SEQ ID NO. 3; Or, the nucleotide sequence of H1 is SEQ ID NO. 8, and the nucleotide sequence of H2 is SEQ ID NO. 9; Or, the nucleotide sequence of H1 is SEQ ID NO. 11, and the nucleotide sequence of H2 is SEQ ID NO.

12.

2. The detection reagent for detecting AD target Micro RNA according to claim 1, characterized in that: It includes the following components in molar parts: H1 50-75 copies, H2 50 parts, Activator 20 copies, CRISPR / Cas endonucleases 75 copies, gRNA 75 copies, 800 copies of the reporter.

3. The detection reagent for detecting AD target Micro RNA according to claim 1 or 2, characterized in that: The CRISPR / Cas nuclease is selected from Cas12a, Cas13a, and Cas9.

4. The detection reagent for detecting AD target Micro RNA according to claim 1, characterized in that: The nucleotide sequence of H1 is SEQ ID NO. 2, and the nucleotide sequence of H2 is SEQ ID NO.

3.

5. The detection reagent for detecting AD target Micro RNA according to claim 1, characterized in that: The nucleotide sequence of gRNA is SEQ ID NO. 4; And / or, the activator is composed of nucleic acids having a nucleotide sequence of SEQ ID NO. 5 and a nucleotide sequence of SEQ ID NO. 6; And / or, the nucleotide sequence of the reporter molecule is TTATT, and both ends of the reporter molecule are modified with a fluorescent group and a fluorescent quenching group, respectively; And / or, H1, H2, and the activator are prepared by dissolving nucleic acid in a buffer solution and subjecting the nucleic acid to thermal annealing treatment; And / or, the detection reagent further comprises 36-50% buffer, the components of the buffer comprising 36.65-50 mM Tris-HCl, 73.3-100 mM NaCl, 7.33-10 mM MgCl2, 73.3-100 μg / mL BSA, 7.33-10 μg / mL heparin sodium, pH 7.5-8.

0.

6. Use of the detection reagent for detecting AD target Micro RNA according to any one of claims 1 to 5, characterized in that: Use of the microfluidic chip in preparing a detection kit or microRNA in the serum of patients with Alzheimer's disease or cancer.

7. The use of the detection reagent for detecting AD target Micro RNA according to claim 6, characterized in that: The method for using the detection kit for MicroRNA in the serum of patients with Alzheimer's disease or cancer comprises the following steps: The test solution is mixed with the detection reagent in the detection kit for detecting MicroRNA in the serum of patients with Alzheimer's disease or cancer, incubated at 37° C. for 30-180 minutes, and the fluorescence signal is detected.

8. A detection kit, characterized in that: A detection reagent for detecting AD target MicroRNA according to any one of claims 1 to 5, or a lipid sphere containing the detection reagent for detecting AD target MicroRNA according to any one of claims 1 to 5; In the lipid sphere containing the detection reagent for detecting AD target Micro RNA according to any one of claims 1 to 5, the dosage ratio of the detection reagent for detecting AD target Micro RNA to the lipid sphere is 1-2 mL: 23-46 mg.

9. The detection kit according to claim 8, characterized in that: The lipid sphere containing the detection reagent for detecting AD target Micro RNA according to any one of claims 1 to 5 is prepared according to the following steps: the detection reagent for detecting AD target Micro RNA is mixed with the lipid sphere.

10. The detection kit according to claim 8, characterized in that The lipid sphere is prepared according to the following steps: cationic lipid molecules, amphiphilic phospholipid molecules and cholesterol are mixed, rotary evaporated to obtain a lipid film, and then incubated in an aqueous solution and extruded to obtain a lipid film; And / or, the mixing is incubated at 37° C. for 6-12 h.

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